The present invention relates to nucleotide sequences of coryneform bacteria, coding for proteins involved in the bio-synthesis of L-serine and to an improved method for the microbial production of L-serine and to a genetically modified microorganism suitable therefor. The present invention further comprises the use of L-serine and/or secondary products thereof in the food, animal feed and/or pharmaceutical industries and/or in human medicine.
In recent years, amino acids such as L-glutamate, L-lysine or branched-chain L-amino acids have increasingly become the focus of economic interest. This is equally true for the amino acid L-serine which serves not only as a precursor for the synthesis of the aliphatic amino acids L-glycine or L-cysteine but also for the production of L-tryptophan from indole and L-serine. To this end, the amino acid L-serine is regarded to have an increasingly economic potential, in particular in the food, animal feed and pharmaceutical industries and also in many areas of human medicine.
Numerous methods for the microbial production of L-serine have been described in the literature. In addition, fermentations of coryneform bacteria for the production of L-serine are already known. Thus, for example, a Corynebacterium glyciniphilum strain is capable of forming L-serine from glycine and carbohydrates (Kubota K., Kageyama K., Shiro T., and Okumura S., (1971), Journal of General Applications in Microbiology, 17: 167–168; Kubota K., Kageyama K., Maeyashiki I., Yamada K., and Okumura S., (1972), Journal of General Applications in Microbiology 18: 365). Here, the enzyme L-serine hydroxymethyltransferase is involved in converting glycine to L-serine (Kubota K. and Yokozeki K., (1989), Journal of Fermentation and Bioengineering, 67(6): 387–390). Furthermore, the bacterial strains used exhibit a reduced serine degradation which can be attributed to a reduction in the activity of the enzyme L-serine dehydratase (Kubota K., Kageyama K., Shiro T. and Okumura S., (1971) Journal of General Applications in Microbiology, 17: 167–168; Kubota K., (1985), Agricultural Biological Chemistry, 49: 7–12).
Furthermore, the fermentative production of L-serine from methanol and glycine with the aid of methylotrophic bacteria, for example of the genus Hyphomicrobium, is described in Izumi Y., Yoshida T., Miyazaki S. S., Mitsunaga T., Ohshiro T., Shiamo M., Miyata A. and Tanabe T., (1993), Applied Microbiology and Biotechnology, 39: 427–432.
In the aforementioned cases, however, formation of the amino acid L-serine starting from carbohydrates requires the addition of the amino acid glycine as a precursor.
Furthermore, methods for fermentation of coryneform bacteria which are capable of producing L-serine directly from carbohydrates without addition of further precursors are already known. Thus, for example, Yoshida H. and Nakayama K., (1974), Nihon-Nogei-Kagakukaishi 48: 201–208 describe bacterial strains of the genus Corynebacterium and, in particular, of the species Corynebacterium glutamicum, which have been obtained by random mutagenesis and which are distinguished, inter alia, by a resistance to the L-serine analogs serine hydroxamate and β-chloroalanine. This causes, inter alia, the metabolic flow to be able to flow increasingly in the direction of L-serine biosynthesis, since the activity of the corresponding enzymes is less inhibited by the final product.
EP 0 931 833 discloses bacterial strains of the species Brevibacterium flavum which were likewise obtained by random mutagenesis and which therefore have defective serine degradation. In addition, the strains described there have a modified serA gene which codes for a feedback-insensitive 3-phosphoglycerate dehydrogenase. These strains additionally contain the genes serB and serC which originate from the heterologous organism Escherichia coli and which code for the enzymes phosphoserine phosphatase and phosphoserine aminotransferase, respectively. The system described here thus has a high complexity with respect to the multiplicity of additionally introduced, partially heterologous, gene structures, combined with a genetic uncertainty of the bacterial strains with respect to the initially mentioned random mutagenesis. This holds the risk of a relatively high instability of such bacterial strains during the course of a large-scale production process. It has further been described that the bacterial strains illustrated here only achieve an increase in L-serine production by a factor of 2 to 5, at most. The reason for this may be, inter alia, a suboptimal expression of heterologous genes.
Another disadvantage of heterologous systems is the low acceptance of foreign DNA-containing systems, in particular for the production of medically and pharmacologically relevant substances and of substances relevant to food.
Besides the biosynthesis of economically interesting L-amino acids such as, for example, L-serine, secretion of these metabolic products into the culture medium is also crucially important for the yield of L-serine in the final product. This export may be unspecific due to diffusion or actively mediated by membrane transport systems, as described, for example, for the amino acids L-isoleucine or L-lysine (Zittrich S. et al., 1994, Journal of Bacteriology, 176: 6892–6899 and Broer S. et al., 1991, European Journal of Biochemistry, 202: 131–153). A problem of those active transport systems is that the capacity of these “export carriers” is quickly exceeded, as soon as the contents of the metabolic product to be transported in the cell exceeds a threshold of the naturally present concentration. This means that, for example in the case of an increased biosynthesis of L-serine, the export thereof out of the cell may be limited.
Consequently, the availability of the genes from coryneform bacteria, which are crucially involved in the biosynthesis of L-serine, for expression in a homologous system is desirable, as is an improved secretion of the L-serine formed into the culture medium.
It is therefore an object of the present invention to provide a system which no longer has the aforementioned disadvantages and makes possible an improved production of L-serine or of metabolic products derivable therefrom and the isolation thereof.